| Linoleic acid( LA, C18:2△9,12 ) and α-linolenic acid (ALA,C18:3△9,12,15 ) havemany necessary sanitarian functions. They can not be synthesized in human beingand vertebrates, and must be got from ingestion. So they both go by the name ofessential fatty acids. They respectively belong to n-6 and n-3 polyunsaturated fattyacids (PUFAs). LA can become γ-linolenic acid (GLA), arachidonic acid (AA) andsome other n-6 PUFAs through a series of desaturation and lengthening reactions.ALA can become EPA, DHA and some other n-3 PUFAs. The functions of n-6 andn-3 PUFAs are often contrary in many aspects. They assort and restrict with eachother to accommodate life activities. So the meal balance of n-6/n-3PUFAs is veryimportant. But in fact the ratio of n-6/n-3 PUFAs in people's meal is generally badlyon the high side. n-6 PUFAs are relatively excessive, but n-3 PUFAs badly lack.This complexion will lead to increases of high thrombus, atheroma and cancer, and italso can aggravate inflammation. So the direction of lipid research should aim todecrease the ratio of n-6/n-3PUFAs, which will make oil healthy. In molecularbiology the PUFAs research work aims to the synthesizing enzymes of PUFAs.△12 fatty acid desaturase (?12 FAD) is the key enzyme to synthesize LA, it cancatalyze oleic acid (C18:1?9) to LA by desaturation. Arachis hypogaea L.(peanut) isthe fourth widest oil plants. In it's seed oil there contains 15%-43% LA, but no n-3PUFAs. Two peanut fatty acid desaturases genes (AhFAD2A,AhFAD2B) have beeencloned, but their functions haven't been studied intensively. ω -3 fatty aciddesaturase(ω-3 FAD) can catalyze LA to ALA. Seed oil of Glycine max(soybean)contains a high level of ALA. Bilyeu KD and his fellows have cloned three soybeanω-3FAD. In developing seeds, the expression of the GmFAD3A come to 60% of thehousekeeping gene expression level while GmFAD3C is 0.2% and GmFAD3Bexpression was below the experimental detection limit. And they find GmFAD3A isthe main contributor for ALA in soybean seed oil, the proffer level is three times highthan that of GmFAD3C. But the functions of the two genes havn't been validated atfirst hand. Saccharomyces cerevisiae(yeast) is used as a preferential host tofunctionally validate fatty acid desaturases in ER, because in yeast there are electrontransferring system and appropriate membrane environment needed by this kind ofdesaturases.To investigate the functions of peanut ?12 fatty acid desaturase (?12 FAD) andsoybean ω-3FAD, the cDNA of AhFAD2B, GmFAD3A and GmFAD3C wereseparately amplified by RT-PCR from peanut and soybean immature seeds, thencloned into the shuttle expression vector p416 to generate the recombinant vectorp4AFAD2B, p4GFAD3A and p4GFAD3C. The resulting vector was transformed intoyeast K601 throuth LiAc method. The positive clones were screened on the CM(Ura-)medium and identified by PCR, and then the recombinant srains Kp4AFAD2B,Kp4GFAD3A and Kp4GFAD3C were obtained. Kp4AFAD2B was cultured inCM(Ura-) liquid medium in 20oC for three days, and Kp4GFAD3A and Kp4GFAD3Cwere cultured in the same way except some exogenous LA added to the medium. Theintracellular fatty acid compositions of all the engineering strains were analyzed bygas chromatography (GC). Kp416 only containing empty p416 plasmid was alsodetected as a comparison sample. The results showed that all the three genes can beexpressed in yeast K601. Kp4AFAD2B produced two new fatty acids namelyhexadecadienoic acid (C16:2?9,12)and C18:2?9,12. The contents of C16:2?9,12andC18:2?9,12 correspondingly reached to 2.1% and 9.2%of the total fatty acidsrespectively in Kp4AFAD2B strain, in the meanwhile,by contrast with control thecontents of palmitoleic acid (C16:1?9) and oleic acid (C18:1?9) correspondinglydecreased. It is suggested that the ?12 FAD encoded by AhFAD2B can not onlycatalyze C18:1?9substrate into C18:2?9,12,but also catalyze C16:1?9 into C16:2?9,12throuth taking off hydrogen atoms at ?12 site. The desaturation rates of C16:1?9和C18:1?9 reached 5.5% and 26.7% respectively. It seems that the enzyme prefersC18:1?9 much more to C16:1?9. Both Kp4GFAD3A and Kp4GFAD3C produced thenew fatty acid of ALA. The content of ALA reached 0.77% and 4.13% of the totalfatty acid separately. Compared with the control strain of Kp416, the content of LAcorrespondingly decreased. It was suggested that both the proteins encoded byGmFAD3A and GmFAD3C can specifically catalyze 18 carbon PUFA substrate of LAinto ALA by taking off hydrogen atoms at ?15location. The desaturation rates of LA inKp4GFAD3A and Kp4GFAD3C reached 6.6% and 34.5% respectively.Thus theresult probably means the GmFAD3C enzyme is much more activated thanGmFAD3A.In this study , the biologic function of a peanut ?12 Fatty acid desaturase wasstudied by heterologous expression in yeast, which built a foundation to cultivate lowLA peanut according with people's health. we also expressed two Glycine max ω-3fatty acid desaturase genes in yeast, the expression is quite efficient. The two geneswere proved to encode active ω -3FAD. An efficient and economical yeastexpressing system(K601-p416 system) which is suitable for the expression of FADwas built, which offered a new investigation for the expression of fatty acid desaturasegenes in yeast.
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